Clustered specific DNA binding sites for an array of activating transcription factors, plus proteins that bend DNA to facilitate contact between bound proteins, have been documented for a number of vertebrate genes (15, 21, 25, 37). These composite structures have been called enhanceosomes (8). The TCR-(15) and the IFN-(25) enhanceosomes, which are assembled in response to dimerization of the T cell receptor or double-stranded RNA, have been most thoroughly explored. Two classes of genes that are very likely dependent upon enhanceosome assembly have received great attention: genes expressed in a tissue-specific manner that acquire multiple binding proteins during development, and genes that are acutely activated by an external stimulus. These latter structures hold appeal for study because they can be examined in cultured cells where induced synchronous changes occur in all the cells under observation, allowing the acute assembly and disassembly of proteins in an enhanceosome to be potentially revealed.
The Stat family of transcription factors (Darnell, 1997; Stark et al., 1998; U.S. application Ser. No. 08/212,185, filed Mar. 11, 1994 and U.S. Pat. No. 5,716,622; all of the foregoing incorporated herein by reference in their entireties) is activated by polypeptide ligands attaching to specific cell surface receptors, and after tyrosine phosphorylation, dimerization and translocation to the nucleus, can participate within minutes in gene activation (11). It seems likely that Stat molecules bind DNA regions where pre-enhanceosome structures exist (26, 27) and that the arrival of activated Stat dimer(s) is key to forming an active enhanceosome (27). Such a possibility is suggested by experiments showing closely spaced binding sites for Stats and other proteins in the response elements for a number of genes (17, 24, 27, 41). Furthermore DNase and permanganate treatment of cell nuclei revealed proteins bound at or near Stat1 sites before polypeptide treatment. This was followed by detection of Stat molecules binding close to the same DNA regions after induction (26).
One intensively studied set of physiologically important genes that are transcriptionally induced in the liver are the “acute phase response proteins” which increase in the wake of bacterial infections and other toxic assaults. IL-6 stimulation of hepatocytes, via the activation of Stat3, is thought to be the main trigger for inducing the acute phase genes (18). One of the best studied enhancers for acute phase response genes is that of the α2-macroglobulin enhancer [(20), reviewed in (18)], a DNA fragment 100 bases long with binding sites for both Stat3 (also called GAS site) and for AP-1, which includes members of the Fos, Jun and ATF families of transcription factors. Extracts from liver nuclei of IL-6 treated animals or transformed hepatocytes (hepatoma cells) in culture indicated induced binding to this region. Since Stat3 and c-Jun interacted in yeast 2-hybrid assays and cooperated in maximizing the transcription responses of reporter genes containing the ˜100 bp enhancer (30, 31), it seemed likely that this genomic region might form a Stat-dependent enhanceosome.
It is towards identifying particular regions of transcription factor interactions responsible for transcriptional activation, and the use of this information in the design of methods and the subsequent identification of agents capable of modulation the interaction, that the present invention is directed.